Use of epineural sheath grafts for neural regeneration and protection

ABSTRACT

Described herein is conduit material that causes minimal inflammatory reaction, and serves as a structural guide for regenerating nerve tissue (e.g., axons). Thus, the invention is directed to methods of treating a nerve injury in an individual in need thereof. The methods employ an isolated, naturally occurring epineural sheath, and can be used, for example, to regenerate nerve tissue in an individual in need thereof. Also provided herein is a device for harvesting an epineural sheath.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of co-pending U.S.application Ser. No. 12/935,232, filed Dec. 8, 2010, which is a NationalStage Application of International Application No. PCT/US2009/039258,filed Apr. 2, 2009, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/123,026, filed Apr. 4, 2008, all of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Severe nerve injuries may result in significant gaps and surgicalmanagement of nerve defects is still a major challenge. When directrepair of the injured nerve with epineural sutures is impossible, thedefect between the nerve stumps has to be bridged by a conduit of somekind, which will facilitate axonal regeneration towards the distal nervestump. Currently, the microsurgical techniques used for the repair ofperipheral nerve defects do not always result in optimal functionaloutcome. The most reliable and widely used method is bridging the defectby an autograft, which is considered the gold standard for nerve gaprepair (Watchmaker, G P et al., Clin. Plast. Surg, 24:63-73 (1997);Siemionow, M., et al., Ann. Plast. Surg., 52:72-79 (2004)). Autograftscan be obtained from functionally less important cutaneous sensorynerves such as a sural nerve, medial antebrachial cutaneous nerve,saphenous nerve, superficial radial nerve and lateral antebrachialcutaneous nerve, which are associated with some donor site morbiditysuch as scarring, anesthesia, neuroma formation (Brandt, J., et al., J.Hand Surg. [Br], 24:284-290 (1999); MacKinnon, S E, Ann. Plast. Surg.,22:257-273 (1989)). There is also a limited source of donor nervesavailable that makes reconstruction of large nerve defects difficult.Nerve allografts have been studied experimentally and used clinically,however the major disadvantage is the need for immunosuppression toprevent rejection (Mackinnon, S E., Plast. Reconstr. Surg. 107:1419-1429(2001); Fox, I K, Muscle Nerve, 31:59-69 (2005)). Therefore nerveallografts are used only in selected cases and are not the primarychoice of peripheral nerve repair in clinical practice.

Various natural and synthetic conduit materials have been investigatedas an alternative technique to nerve autografts. Vein grafts, collagen,muscle, denaturated muscle basal lamina, tendon, mesothelium, amnion andepineural sheaths are the natural conduits investigated by researchers(Tang, J B, J. Reconstr. Microsurg., 11:21-26 (1995); Mohammad, J.,Plast. Reconstr. Surg., 105:660-666 (2000); Chen, L E, J. Reconstr.Surg., 10:137-144 (1994); Atabay, K., et al., Plast. Surg. Forum, 18:121(1995); Siemionow, M., et al., Ann. Plast. Surg., 48:281-285 (2002);Tetik, C., et al., Ann. Plast. Surg., 49:397-403 (2002); Ayhan, S., etal., J. Reconstr. Microsurg., 16:371-378 (2000); Yavuzer, R., et al.,Ann Plast. Surg., 48:392-400 (2002); Lundborg, G., et al., J Hand Surg.[Am], 7:580-587 (1982); Archibald, S J., et al., J. Comp. Neurol.,306:685-696 (1991); Keskin, M., et al., Plast, Reconstr. Surg.,113:1372-1379 (2004)). Synthetic materials for bridging the defect havegained attention and silicon tubes, polylactic acid, polyglycolic acid,poly-3-hydroxybutyrate, polyurethane, and poly(organo)phosphazene tubeswere used in experimental and clinical studies with encouraging results(Hudson, T W, et al., Clin. plast. Surg., 26:485-497 (1999); Langone,F., et al., Biomaterails, 16:347-353 (1995); Lundborg, G., et al., Exp.Neurol., 76:361-375 (1982); Weber, R A., et al., Plast. Reconstr. Surg,106:1036-1045 (2000); Hazari, A., et. al., Br. J. Plast. Surg.,52:653-657 (1999); Navissano, M., et al., Micorsurgery, 25:268-271(2005); Nalamura, T., et al., Brain Res., 1027:18-29 (2004)). Theseconduits are used as tubulized chambers and present with differentadvantages, but also disadvantages such as inflammation, foreign bodyreaction, compression, and toxicity of degradation products.

Alternative methods and compositions with less morbidity for treatingnerve injuries are needed.

BRIEF SUMMARY OF THE INVENTION

Described herein is conduit material that causes minimal inflammatoryreaction, and can serve as a structural guide for regenerating, or as ashield for protecting, nerve tissue (e.g., axons). Thus, the inventionis directed to methods of treating an injury to a (one or more) nerve orprotecting a nerve in an individual in need thereof. The methods employall or a portion of an isolated, naturally occurring epineural sheath,and can be used, for example, to regenerate nerve tissue in anindividual in need thereof.

In one embodiment, the invention is directed to a method of repairing anerve gap having a proximal nerve stump and a distal nerve stump in anindividual in need thereof. The method comprises attaching all or aportion of an (one or more) isolated, naturally occurring epineuralsheath (e.g., in the form of a tube, a flat sheath, a strip, a patch, acord, a scaffold, a paste or a powder) to the proximal nerve stump andto the distal nerve stump, thereby producing a nerve graft. The nervegraft is maintained under conditions in which nerve tissue isregenerated between the proximal nerve stump and the distal nerve stump,thereby repairing the nerve gap.

In a particular embodiment, the invention is directed to a method ofrepairing a nerve gap having a proximal nerve stump and a distal nervestump in an individual in need thereof, comprising attaching anisolated, naturally occurring epineural tube to the proximal nerve stumpand to the distal nerve stump, thereby producing a nerve graft. Thenerve graft is maintained under conditions in which nerve tissue isregenerated between the proximal nerve stump and the distal nerve stump,thereby repairing the nerve gap.

In a particular embodiment, the invention is directed to a method ofrepairing a nerve gap having a proximal nerve stump and a distal nervestump in an individual in need thereof, comprising attaching a flatepineural sheath (e.g., a full rectangular epineural sheath) to theproximal nerve stump and to the distal nerve stump, thereby producing anerve graft. The nerve graft is maintained under conditions in whichnerve tissue is regenerated between the proximal nerve stump and thedistal nerve stump, thereby repairing the nerve gap.

In another embodiment, the invention is directed to a method ofrepairing a nerve gap having a proximal nerve stump and a distal nervestump in an individual in need thereof, comprising attaching at leastone epineural strip (a first epineural strip, a second epineural strip,a third epineural strip, a fourth epineural strip, etc.) to the proximalnerve stump and to the distal nerve stump, thereby producing a nervegraft. The nerve graft is maintained under conditions in which nervetissue is regenerated between the proximal nerve stump and the distalnerve stump, thereby repairing the nerve gap. The method can furthercomprise splitting an epineural sheath longitudinally, thereby producinga first epineural strip, a second epineural strip, a third epineuralstrip, etc. prior to attaching the first epineural strip, the secondepineural strip, the third epineural strip, the fourth epineural stripetc. to the nerve stump.

In a particular embodiment, the invention is directed to a method ofrepairing a nerve gap having a proximal nerve stump and a distal nervestump in an individual in need thereof, comprising attaching a firstepineural strip to the proximal nerve stump and to the distal nervestump, and attaching a second epineural strip to the proximal nervestump and to the distal nerve stump, thereby producing a nerve graft.The nerve graft is maintained under conditions in which nerve tissue isregenerated between the proximal nerve stump and the distal nerve stump,thereby repairing the nerve gap. The method can further comprisesplitting an epineural sheath longitudinally, thereby producing a firstepineural strip and a second epineural strip, prior to attaching thefirst epineural strip and the second epineural strip to the nerve stump.

The invention is also directed to a method of protecting neural tissuein an individual in need thereof, comprising attaching an isolated,naturally occurring epineural sheath to the neural tissue (e.g., adorsal root ganglion, spinal cord), thereby covering the neural tissueand maintaining the neural tissue under conditions in which neuraltissue is isolated, thereby protecting the neural tissue. In oneembodiment, the neural tissue is injured neural tissue. In anotherembodiment, the injured neural tissue produces neuropathic pain in theindividual. In yet another embodiment, the injured neural tissue iscompressed. In particular embodiments, the neural regeneration occurs.

A device for harvesting (isolating) an epineural sheath is also providedherein. The device for harvesting an epineural sheath comprises a hollowtube having a distal end and a proximal end, wherein the distal endcomprises a forwardly curved protrusion extending from a side wall ofthe tube and curving radially inward to a central line of the tube, andthe proximal end comprises a flange. In one embodiment, the hollow tubeis rotatably coupled to a rotating drive. In another embodiment, thedevice further comprises an irrigation system in fluid communicationwith the hollow tube. The irrigation system can be in fluidcommunication with the hollow tube using, for example, flexible tubing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F are schematic representations of one embodiment of themethods described herein. The preparation of the rectangular epineuralsheath graft (FIGS. 1A, 1B and 1C). Repair of the defect with one strip(FIG. 1D), two-strips (FIG. 1E) and full rectangular epineural sheathgraft (FIG. 1F).

FIGS. 2A-2E are photographs showing creation of the nerve defect (FIGS.2A and 2B). The epineurium is incised with scissor (FIGS. 2C and 2D) andflat rectangular shaped epineural sheath graft is obtained (FIG. 2E).

FIGS. 3A-3C are photographs showing the appearance after the repair ofthe nerve defect with one-strip (FIG. 3A), two-strip (FIG. 3B) and fullrectangular epineural sheath graft (FIG. 3C)

FIG. 4 is a photograph showing full recovery at toe-spread at 12 weeksfrom a rat from full sheath repair group

FIG. 5 is a graph showing P1 and N2 latencies obtained by SSEP test inanimal repaired with full epineural sheath showing similar waveformpatterns. While P1 values were 15.83 ms in the operated side (superiorwave) and 15.81 ms in the ipsilateral control limb (inferior wave), N2latencies were 24.02 ms and 22.66 ms respectively.

FIGS. 6A-6C are photographs showing gastrocnemius muscles from thenon-operated site and operated sites at 12 weeks harvested fromone-strip (FIG. 6A), two-strip (FIG. 6B), and full sheath repair group(FIG. 6C). The muscles in the left sides are from non-operated limb andin the right side are muscles from operated limb. Macroscopic atrophywas minimal at the operated site at rats repaired with full rectangularepineural sheath graft.

FIGS. 7A-7C are photographs showing the appearance of the grafts fromone-strip group (FIG. 7A), two-strip group (FIG. 7B) and fullrectangular graft group (FIG. 7C) at 12th weeks prior to harvesting.Only a fibrotic band was observed in one-strip repair group but normalnerve structure was seen at animals repaired with full rectangularsheath graft.

FIGS. 8A-8B are photographs showing (FIG. 8A) a cross section of newlyformed nerve in full sheath repair group. Normal nerve structure isobserved with outer epineural sheath; and (FIG. 8B) the vascularizationof the nerve at the distal repair site from the distal towards thegraft.

FIGS. 9A-9C show the analysis of the toluidine-blue stained sectionsfrom the grafts in animals of (FIG. 9A) Group 5 (full sheath) and (FIG.9B) of group 4 (two-strip) showing regenerating nerve fibers atpostoperative week 12 (magnification.times.400). (FIG. 9C) Section fromgraft in group 3 (one-strip) showing paucity of regenerating axons(magnification.times.400)

FIG. 10 is a series of graphs showing histomorphometric results.

FIG. 11 is a schematic showing the harvest of an epineural tube.

FIG. 12 is a schematic of harvesting of an epineural tube and bonemarrow stromal cell (BMSC) preparation.

FIG. 13 shows the histology results twelve weeks after rat sciatic nerverepair; Group A: saline injection; Group B: Isogenic BMSCs; Group C:Allogenic BMSCs. Isogenic BMSCs showed higher number of regeneratedaxons (90.6.+−0.26.9) compared to Group A (71.4.+−0.3.0) and Group C(76.4.+−0.5.4).

FIG. 14A shows NGF staining 6 weeks after epineural tube transplantsupported with isogenic stromal cells, and shows that transplanted cellsPKH26 labeled cells are associated with neural marker expression.

FIG. 14B shows staining at 18 weeks in the distal part of the tube whichconfirms nerve regeneration over the entire nerve segment from theproximal end to the distal end.

FIG. 15 shows immunostaining results showing laminin B expression in theepineural tube.

FIG. 16 shows immunostaining results showing laminin, GFAP, VEGF, S-100,NGF and MHC class II expression in the epineural tube.

FIG. 17 shows immunostaining and peroxidase staining showing adifference between normal epineural sheath without VEGF expression andepineural sheath patch 72 hours after removal from the dorsal rootganglion indicating VEGF expression and confirming neovascularizationpotential of the epineural patch.

FIG. 18 shows an epineural sheath harvesting device that can be usedwith a pump operated nerve harvesting system.

FIG. 19 shows a pump operated epineural sheath harvesting system for useas an epineural tube recovery device.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are methods of treating a neural injury (e.g., an injuryto a peripheral nerve; an injury to a cranial nerve; injury to thespinal cord) in an individual in need thereof. In particularembodiments, the invention provides methods of repairing and/orprotecting nerve tissue in an individual in need thereof, comprisingattaching all or a portion of an (one or more) isolated, naturallyoccurring epineural sheath to the nerve tissue in need of repair orprotection. In some instances, all or a portion of the (one or more)isolated, naturally occurring epineural sheath is used to repair a nerve(e.g., as a nerve graft to repair a nerve gap). In other instances, allor a portion of the isolated, naturally occurring epineural sheath isused as to cover or shield a neural tissue (e.g., as an epineural patchto cover dura in the brain (e.g., cerebellum); as a nerve patch cover anintact nerve that is damaged). An intact nerve or nerve root can bedamaged, for example, due to inflammation such as in the case oflaminectomy, after surgical intervention of decompression in diabeticand non-diabetic neuropathies, after neuroma resection, etc.

The nerve graft and/or nerve patch is maintained under conditions inwhich the nerve tissue is repaired and/or protected. The methods can beused, for example, to regenerate nerve tissue in an individual in needthereof.

In a particular embodiment, the invention provides for a method ofrepairing a nerve gap having a proximal nerve stump and a distal nervestump in an individual in need thereof. In the method, an isolated,naturally occurring epineural sheath is attached to the proximal nervestump and to the distal nerve stump, thereby producing a nerve graft.The nerve graft is maintained under conditions in which nerve tissue isregenerated between the proximal nerve stump and the distal nerve stump,thereby repairing the nerve gap.

Typically, nerve fibers are wrapped in a connective tissue called theendoneurium. Groups of fibers surrounded by their endoneurium arearranged in bundles called fascicles, and each fascicle is wrapped inconnective tissue called the perineurium. The outermost covering aroundthe entire nerve is the epineurium. As used herein an (one or more)“epineural sheath” is an (one or more) epineurium of a (one or more)nerve. As described herein, an isolated, naturally occurring epineuriumsheath is used in the methods of the invention. A “naturally occurring”epineural sheath refers to an epineural sheath obtained from naturalsources; that is, an epineural sheath that is not synthetic(non-synthetic). Epineural sheaths that are “isolated”, include pure(essentially pure) epineural sheaths, that have been separated away frommolecules and other tissues (e.g., endoneurium, perineurium, fasicles,blood components, inflammatory molecules) of their source of origin(e.g., an individual; an isolated nerve), and include epineural sheathsobtained by methods described herein or other suitable methods.

The epineural sheath can be obtained from a variety of nerves, such asnerves from invertebrates, vertebrates or a combination thereof. In oneembodiment, the naturally occurring, isolated epineural sheath isobtained from (isolated from) a mammalian nerve such as a nerve ofprimate (e.g., human), porcine, canine, feline, bovine, and/or murineorigin. In other embodiments, the epinueral sheath is an autologousepineural sheath, an allogenic epineural sheath, an isogenic epineuralsheath, a xenogenic epineural sheath or a combination thereof. In aparticular embodiment, the epineural sheath is obtained from a cadaver(e.g., a human cadaver).

In addition, the epineural sheath can be obtained from a variety of typeof nerves, such as from a sensory nerve and/or a motor nerve. Forexample, although not necessary, in embodiments in which the methods areused to repair a nerve gap in a sensory nerve, the epineural sheath canbe obtained from a sensory nerve (e.g., from a sensory nerve that is thesame as, similar to or different from, the sensory nerve that is beingrepaired); and in embodiments in which the methods are used to repair anerve gap in a motor nerve, the epineural sheath is obtained from amotor nerve (e.g., from a motor nerve that is the same as, similar to ordifferent from, the motor nerve that is being repaired).

As will be apparent to one of skill in the art, all or a portion of annaturally occurring, isolated epineural sheath can take a variety ofshapes for use in the methods of the invention, and the shape willdepend upon a variety of factors, such as the properties of the nervethat is to be repaired (e.g., nerve type, nerve diameter, nerve length),the type of nerve injury and/or the condition of the individual (e.g.,patient). For example, one or more epineural sheaths can be used as atube (e.g., a tube having two free ends or lumens; a hollow tube), orone or more tubes can be longitudinally split and used as a flatrectangular sheath. In addition, one or more epineural sheaths can beformed into one or more strips, cords (e.g., twisted strips, plain orenriched with cells), patches, scaffolds (e.g., filled with cells,slow-releasing growth factor), pastes, powders (e.g., with a gel),putty(ies) or a combination thereof for use in the methods of theinvention. As will be apparent to one of skill in the art, one or moreof these forms can be achieved using one or more epineural sheaths(e.g., multiple epineural sheaths secured together, e.g., as a largesheet or secured together in multiple layers and filled with powder, geland/or factors that enhance nerve growth and/or regeneration).

In the methods of the invention, all or a portion of a naturallyoccurring, isolated epineural tube can be used. In one embodiment, oneor more naturally occurring, isolated epineural tubes can be used in themethods. In another embodiment, one or more naturally occurring,isolated epineural tubes can be split (e.g., longitudinally) and used asa (e.g., flat) rectangular sheath in the methods. In embodiments inwhich two or more naturally occurring, isolated epineural tubes aresplit longitudinally thereby producing two or more rectangular sheaths,the two or more rectangular sheaths can be used to make a largerectangular sheath or placed in layers. In yet another embodiment, theepineural tube can be split (e.g., longitudinally) into one or morestrips, and the epineural strips can be used in the methods describedherein.

Accordingly, the invention is directed to a method of repairing a nervegap having a proximal nerve stump and a distal nerve stump in anindividual in need thereof, comprising attaching an isolated, naturallyoccurring epineural tube to the proximal nerve stump and to the distalnerve stump, thereby producing a nerve graft (e.g., a tubular nervegraft). The nerve graft is maintained under conditions in which nervetissue is regenerated between the proximal nerve stump and the distalnerve stump, thereby repairing the nerve gap

In another embodiment, the invention is directed to method of repairinga nerve gap having a proximal nerve stump and a distal nerve stump in anindividual in need thereof, comprising attaching a flat epineural sheathto the proximal nerve stump and to the distal nerve stump, therebyproducing a nerve graft. The nerve graft is maintained under conditionsin which nerve tissue is regenerated between the proximal nerve stumpand the distal nerve stump, thereby repairing the nerve gap.

In yet another embodiment, the invention is directed to a method ofrepairing a nerve gap having a proximal nerve stump and a distal nervestump in an individual in need thereof, comprising attaching at leastone epineural strip (multiple strips, such as a first epineural strip, asecond epineural strip, a third epineural strip, a fourth epineuralstrip, etc.) to the proximal nerve stump and to the distal nerve stump,thereby producing a nerve graft. The nerve graft is maintained underconditions in which nerve tissue is regenerated between the proximalnerve stump and the distal nerve stump, thereby repairing the nerve gap.

The method can further comprise splitting an epineural sheathlongitudinally, thereby producing one or more epineural strips (multiplestrips, such as a first epineural strip, a second epineural strip, athird epineural strip, etc.) prior to attaching the one or moreepineural strips to the nerve stump. For example, the method can furthercomprise splitting an epineural tube longitudinally into a firstepineural strip and a second epineural strip, prior to attaching thefirst epineural strip and the second epineural strip to the nerve stump.

In a particular embodiment, the invention is directed to a method ofrepairing a nerve gap having a proximal nerve stump and a distal nervestump in an individual in need thereof, comprising attaching a firstepineural strip to the proximal nerve stump and to the distal nervestump, and attaching a second epineural strip to the proximal nervestump and to the distal nerve stump, thereby producing a nerve graft.The nerve graft is maintained under conditions in which nerve tissue isregenerated between the proximal nerve stump and the distal nerve stump,thereby repairing the nerve gap.

In particular embodiments of the invention, the length of the defectbetween the proximal and distal nerve stump is assessed. The epineuralsheath (e.g., an epineural tube segment) will match the size of thedefect (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 cm of length) between the proximal and distal nervestump, and the diameter of the proximal and distal nerve stump (e.g., 1,2, 3, 5, 10, 15, 20, 25, 30 mm of diameter). In particular embodiments,the epineural sheath can match the type of nerve to which it will beconnected, which can be either purely sensory (e.g., sural nerve),purely motor (e.g. radial nerve), or mixed sensorimotor nerve type(e.g., median nerve). In these embodiments, the sensory tube willconnect the sensory nerve stumps, motor tube, motor nerve stumps, andsensorimotor stumps.

In one embodiment, the epineural tube is connected to the proximal (ordistal) nerve stump using microsurgical technique with microsurgicalsutures (4-6 sutures 10/0) or using gluing technique with application ofdifferent types of tissue sealants or glues, or using staple technique,or a combination of the above. The epineural sheath can be overlying,from about 1 mm to 20 mm, about 3 mm to about 15 mm, about 5 mm to about10 mm, and about 7 mm to about 12 mm the proximal (or distal) nervestump, so that the severed proximal (or distal) end of the nerve will beinserted inside the tube. Once the proximal end repair is finished, thesame steps will be followed at the distal (or proximal) nerve end byinserting the distal nerve stump into the distal tube segment. The tubecan be applied for nerve repair, depending on the length of the gap andnerve diameter. It can be applied as an empty tube to replace existingtechnologies for short gaps, and in particular, as a tube filled withthe different types of cells (e.g., Schwann cells, bone marrow cells,mesenchymal stem cells, chimeric cells), or as a tube filled with nervegrowth enhancing factors (e.g., VEGF, NGF growth factor),anti-inflammatory factors, or combination thereof.

The epineural sheath can be used as a patch for a variety ofapplications. For example, the patch can be used to close dural defectsin one of several ways. As a nerve-like and nerve-friendly tissue patch,it can protect neural tissues from inflammatory factors (e.g., cytokinesand chemokines) released by the surrounding environment, and keep neuraltissue friendly factors accessible via a natural system of nerve-bloodbarrier or blood-brain barrier.

Accordingly, the invention is also directed to a method of protectingneural tissue in an individual in need thereof, comprising attaching anisolated, naturally occurring epineural sheath to the neural tissue(e.g., a dorsal root ganglion), thereby covering the neural tissue andmaintaining the neural tissue under conditions in which the neuraltissue is isolated, thereby protecting the neural tissue. In oneembodiment, the neural tissue is injured neural tissue. In anotherembodiment, the injured neural tissue produces neuropathic pain in theindividual. In yet another embodiment, the injured neural tissue iscompressed. In particular embodiments, neural regeneration occurs.

Using standard surgical technique, an epineural patch of sufficient size(e.g., ranging from 1.times.1 cm up to 10.times.10 cm), and thickness(single versus multiple layer patch) which effectively covers and sealsoff the defect can be secured (e.g., using suture) into the desiredposition. The effectiveness of the seal can be augmented with variouscommercially available glues, such as fibrin glue, staplers, and otheradhesive materials. Alternatively, the epineural patch can be placedtopically as an “on lay graft” on the exposed dura relying onmechanical, chemical or electrostatic adhesive forces to preventdislodgment, or can be placed topically on the exposed dura and securedwith commercially available glue, such as fibrin glue. The epineuralpatch can be fashioned in such a manner where part or all of the downside is covered with an adhesive substance, such as fibrin glue. It canthen placed topically on the exposed neural tissues and secured to theunderlying neural tissues by the adhesive properties of the glue. Thisapplication can be augmented with BMSCs or MSCs as an inherent part ofthe graft or BMSCs or MSC's injected under patch surface or betweenpatch layers.

The patch can be also be used to cover exposed neural tissues in one ofseveral ways. In this embodiment, the epineural patch is used as ashielding neuro-like or neuro-friendly tissue which protects injured,decompressed or repaired nerves from forming adhesions with or scarringby non-neural type of tissues in the surroundings such as muscles, bone,tendon, skin. This embodiment helps with nerve gliding after surgery,and protects against nerve exposure to scar tissue formation fromunderlying or overlying tissues. In this embodiment, the epineural patchprotects nerves from becoming adherent to the surrounding non-neuraltissues which prevents nerve from development of nerve traction (againstadhesion or scar) injury.

The epineural patch can be used as single or multiple layer patch, andcan be placed topically on the exposed neural tissues relying onmechanical, chemical or electroadhesive forces to prevent dislodgment.The epineural patch can be placed topically on the exposed neuraltissues and secured with commercially available sutures, staplers andglue, such as fibrin glue. The epineural patch can be fashioned in sucha manner where part or all of the down side is covered with an adhesivesubstance, such as fibrin glue. It is then placed topically on theexposed neural tissues and secured to the underlying neural tissues bythe adhesive properties of the glue or by sutures.

The above applications can be augmented with bone marrow stromal cells(BMSCs) being the inherent part of the graft or BMSCs injected underpatch surface or between patch layers for enhancement of nerveregeneration.

The epineural sheath can also used to inhibit neuroma formation afterneuroma revision surgeries requiring nerve implantation into the muscleor bone tissue. The epineural sheath can also be used to cover thetransected nerve end (after neuroma resection) by inserting the proximalnerve segment into the proximal end of an epineural sheath tube andattaching the tube to the nerve stump (e.g., using a suture, stapler,sealant or glue). Care should be taken to leave enough space within thetube for the nerve stump to settle freely in the tube, thus, thereshould be an excess of the epineural tube at the distal end which willbe sealed, ligated, sutured or left open before implantation of thenerve-tube complex into the muscle or bone. Thus, the epineural sheath(e.g., an epineural sheath tube) can be used to cover nerve stump afterneuroma resection without implantation into the muscle or bone. Thetechnique of covering the neuroma stump is the same as described above.

Compression of the spinal cord and nerve roots can result inirreversible histological and physiological changes such as intraneuralfibrosis, demyelination, and neuronal loss. The epineural sheath can beused as a protective anti-inflammatory sheath or to increase neuralregeneration or vascularization in patients undergoing decompressionprocedures for myelopathy secondary to spondylosis, disc herniation,trauma, tumor, and/or complicated by diabetes. In addition, theepineural sheath can be used as a dura mater substitute in cases of adural deficit, an iatrogenic durotomy and/or a dural transplant. Theepineural sheath can also be used to prevent scarring and adhesions inpatients undergoing decompressive procedures of the spinal cord, thecalsac, and nerve roots and/or suffering from radiculopathy/myelopathy. Theepineural sheath can also be used to increase neuronal regeneration anddecrease inflammation in patients with radiculopathy/myelopathy and tocreate an optimal microenvironment and increase neuronal regeneration inpatients suffering from spinal cord injury.

As is apparent to one of skill in the art, different lengths anddiameters of epineural sheaths may be used in the methods of theinvention, and will depend upon a variety of factors, such as theproperties of the nerve that is to be repaired (e.g., nerve type, nervediameter), the type of neural injury (e.g., the dimensions, such aslength and width, of a nerve gap) and/or the condition of theindividual. In some embodiments, the epineural sheath can be from about1 mm to about 10 cm in length. In other embodiments, the epineuralsheath can be from about 1 cm to about 10 cm in width. For example, theepineural sheath can have different tube diameters (e.g., about 1 mm, 2mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23mm, 24 mm, 25 mm, 26 mm, 27 mm, 28 mm, 29 mm, 30 mm) and lengths (e.g.,about 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm), andpatch sizes (about 1 cm.sup.2.times.100 cm.sup.2 such as 2.times.2 cm,3.times.3 cm, 4.times.4 cm, 5.times.5 cm, 6.times.6 cm 7.times.7 cm,8.times.8 cm, 9.times.9 cm, 10.times.10 cm, 20.times.20 cm, 30.times.30cm, 40.times.40 cm, 50.times.50 cm, 60.times.60 cm, 70.times.70 cm,80.times.80 cm, 90.times.90 cm, 100 cm.times.100 cm, etc). The tubes andpatches can be linked or custom designed (cut from whole sheath etc.).

Methods for obtaining or harvesting isolated, naturally occurringepineural sheaths are provided herein, and are known to those of skillin the art. In addition, preservation methods to reduce immunogenicityfor allografts and to keep stored epineural grafts for off shelf use andbanking following methods are also provided herein. After harvesting,cryopreserved, cold stored, or lyophilized epineural sheaths can be usedas different lengths, sizes, and widths.

Examples of methods for harvesting an isolated, naturally occurringepineural sheath from the sciatic nerve are provided herein. As will beapparent to one of skill in the art, other methods can be used toharvesting an isolated, naturally occurring epineural sheath from othersources using routine skills. In one embodiment, the access to theperipheral nerve (e.g., sciatic nerve) is made by skin incision andsubcutaneous tissue dissection down to the anatomical location of thenerve. At this level the sciatic nerve is cleared of all surroundingtissues by blunt dissection as far proximally as the sacral plexus andas far distally as its division into the terminal nerve branches. Allcollateral branches arising from the sciatic nerve throughout its lengthcan be detached and used separately to create an epineurial sheathtubular grafts of different size diameters and lengths.

At this point the sciatic nerve is ready to be dissected out. The nerveis transected as proximal as is feasible at its origin from the sacralplexus, and then transected distally where the nerve divides into itsterminal components, at the level of insertion into the muscle.

Depending upon the area of nerve harvest, the nerve can then besuspended on either a straight driver/irrigator with round tip (e.g., 30gauge.times.25 mm depending on nerve diameter—the driver diameter istypically smaller than nerve diameter), on a curved/hook finisheddriver/irrigator, or on a screwdriver type of irrigator. The irrigatorcan be filled with chilled solution (either cryopreservation solutionfor long term storage, or nerve culture medium or combination ofboth—depending on the fate of graft) and kept moist on the dissectionboard by soaking it with 0.9% sodium chloride.

Under microscope or loop magnification the axons can then gently beteased from its epineural sheath with the use of circular motion ofdriver/irrigator and jeweler fine forceps pulling the sheath away fromthe axons and driver in the “devaginating maneuver”, so that the axonfibers are pulled from the distal end whilst the epineural sheath isheld from the proximal end on the driver/irrigator. Once all axons, theperineurium and the endoneurium are removed the intact, clear epineuralsheath can be irrigated and left as a product of this process and isthen inspected for integrity.

Accordingly, also provided herein is a device for harvesting (isolating)an epineural sheath (e.g., a device for removing neural tissue (e.g.,fascicles, axons) from its epineurium, thereby harvesting an epineuraltube). Example of such a device are provided in FIGS. 18 and 19.

In one embodiment, the device for harvesting an epineural sheathcomprises a hollow tube having a distal end and a proximal end, whereinthe distal end comprises a forwardly curved protrusion extending from aside wall of the tube and curving radially inward (forward) to a centralline of the tube, and the proximal end comprises a flange (e.g.,shoulder). Any type of hollow tube, such as a hollow needle, a hollowcatheter, and a hollow cannula, can be used in the device providedherein. The length and diameter of the hollow tube will depend upon thetype of nerve from which the epineural sheath is being harvested. Forexample, the hollow tube can range in diameter from about 0.1 mm toabout 15 mm. In particular embodiments, the diameter of the hollow tubeis about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7, 0.8 mm,0.9 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11mm, 12 mm, 13 mm, 14 mm, or 15 mm. The hollow tube can range in lengthfrom about 50 mm to about 180 mm. In particular embodiments, the lengthof hollow tube is about 50 mm, 55 mm, 60 mm, 65 mm, 70 mm, 75 mm, 80 mm,85 mm, 90 mm, 95 mm, 100 mm, 105 mm, 110 mm, 115 mm, 120 mm, 125 mm, 130mm, 135 mm, 140 mm, 145 mm, 150 mm, 155 mm, 160 mm, 165 mm, 170 mm, 175mm, or 180 mm.

The curved protrusion (e.g., tip, hook, prong, anchor) at the distal endof the hollow tube is used to engage (e.g., capture, grab) the nervefiber(s) within the epineural sheath. Depending on the type of nervewithin the epineural sheath that is to be removed (e.g., extracted), theprotrusion can be thin, wide, flat, rounded or some combination thereof.

The flange at the distal end of the hollow tube is present to provide,for example, increased thickness and/or strength to the hollow tube. Inaddition the flange can be used to attach additional components to thedevice.

For example, the hollow tube can be rotatably coupled to a rotatingdrive, such as a drill or a pump.

The device can also comprise an irrigation system that is in fluidcommunication with the hollow tube. In one embodiment, the irrigationsystem is an irrigation pump. The irrigation system can be in fluidcommunication with the hollow tube using, for example, flexible tubing.

In a particular embodiment, the device for harvesting an epineuralsheath comprises (a) a hollow tube having a distal end and a proximalend, wherein the distal end comprises a forwardly curved protrusionextending from a side wall of the tube and curving radially inward to acentral line of the tube, and the proximal end comprises a flange, (b) arotating device that is rotatably coupled to the flange of the hollowtube, and (c) an irrigation system that is attached to the rotatingdevice and which is in fluid communication with the hollow tube.

As will be apparent to those of skill in the art, the device can beportable or stationary. As will also be apparent the device can be usedin combination with other devices or instruments such as forceps to pullthe sheath away from the distal end of the device.

Also provided is a method of harvesting (isolating) an epineural sheathusing the device as described herein.

Following harvesting of the epineural sheath, the epineural sheaths canbe stored for later use. For example, following harvesting, theepineural sheaths can be saturated with dimethlyosulfoxide(cryopreserving agent) and frozen (e.g., by computer controlled freezer(−196 degrees Celcius)) and stored in liquid nitrogen. See Example 3 formore details of this embodiment of the invention.

In one embodiment, an acellular tissue matrix can be fractured so as togive a particulate tissue matrix. The acellular tissue matrix can bemade from all or part of cadaver areolar connective tissue with vascularcomponents disposed among randomly and partly longitudinally arrangedbundles of collagen fibrils; and can include collagen fibrils andelastic fibers to which, subsequent to rehydration, viable neural cells,endothelial cells, or viable epithelial cells attach. This tissue matrixcan be in the form of, for example, a patch of variable shape, variablethickness, variable surface area, which can be either single layered ormultilayered, with the option of adding cells or growth factorin-between the layers (“sandwich patch”).

In another embodiment, the method comprises fracturing an cellulartissue matrix so as to give a particulate tissue matrix. The cellulartissue matrix can be made from all or part of cadaver areolar connectivetissue with vascular components disposed among randomly and partlylongitudinally arranged bundles of collagen fibrils; and can includecollagen fibrils and elastic fibers to which viable neural cells,endothelial cells, or viable epithelial cells attach. This tissue matrixcan be in the form of, for example, a patch of variable shape, variablethickness, variable surface area, which can be either single layered ormultilayered, with the option of adding cells or growth factorin-between the layers (“sandwich patch”).

As described herein, the methods of the invention provide for methods ofrepairing and/or protecting nerve tissue in an individual in needthereof. In a particular embodiment, the methods are used to repair anerve gap. As used herein, a “nerve gap” refers to an interruption(partial, complete) of continuity (e.g., a break, a lesion, traumatic oriatrogenic transaction or resection) in a (one or more) nerve orfascicle, which results in a nerve or fascicle having an interruption(partial, complete) of conduction. In particular embodiments, the nervegap is from about 0.5 mm to about 20 cm, from about 1 mm to about 18 cm,from about 2 mm to about 16 cm, about 4 mm to about 14 cm about 6 mm toabout 12 mm and about 8 mm to about 10 cm. As a result of the nerve gap,a nerve having a proximal nerve stump and a distal nerve stump isproduced.

In the methods of the invention, an isolated, naturally occurringepineural sheath is attached to the proximal nerve stump and the distalnerve stump, thereby producing a nerve graft that bridges (spans;connects; links) the proximal nerve stump to the distal nerve stump. Theepineural sheath can be attached to the proximal and distal nerve stumpsusing a variety of methods known to those of skill in the art. Forexample, the epineural sheath can be attached using one or more sutures,glues (e.g., fibrin glue), staples, other adhesive materials or acombination thereof. In a particular embodiment, the epineural sheath isattached to the proximal nerve stump using at least one suture andattached to the distal nerve stump using at least one suture.

The nerve graft produced is maintained under conditions in which nervetissue is regenerated and/or nerve conduction (partial or complete)between the proximal nerve stump and the distal nerve stump is regained.For example, as will be apparent to one of skill in the art, in someembodiments, after introducing the nerve graft in an individual asdescribed herein, the area of the nerve graft (e.g., the surgical area)can be maintained under conditions to (in an environment that) promotehealing, and prevent or minimize scar formation and/or infection, of thearea.

The methods described herein can further comprise contacting theepineural sheath (e.g., filling the graft; coating the sheath) withcells that aid and/or enhance regeneration of neural tissue. Examples ofsuch cells include progenitor cells, stem cells (e.g., mesenchymal stemcells), bone marrow derived cells (e.g., bone marrow stromal cells(BMSC)), mesenchymal stromal cells, dendritic cells, adipose (fat)cells, or a combination thereof (e.g., chimeric cells). The cells can beautologous, allogenic, isogenic, xenogenic or a combination thereof(e.g., chimeric cells). As used herein, a “chimeric cell” refers to acell which is a fusion of one or more autologous, allogenic, or isogeniccells with one or more autologous, allogenic, or isogenic cells. Thefused cells can be the same (e.g., one or more BMSC fused with one ormore BMSC), similar (e.g., one or more BMSC fused with one or moremesemchymal stromal cells) or different (e.g., one or more BMSC fusedwith one or more dendritic cells) type of cell. In a particularembodiment, the chimeric cell is a donor cell (e.g., a donor origin bonemarrow progenitor such as a CD90 cell) fused with a recipient cell whichis the same type of cell as the donor cell (e.g., a donor origin bonemarrow progenitor such as a CD90 cell).

In addition, the methods described herein can further comprisecontacting (e.g., filling the graft; coating the sheath) the epineuralsheath or nerve graft with factors that aid or enhance regeneration ofnerve tissue. Examples of such factors include neurotropic andneurotrophic factors. Specific examples include nerve growth factors(NGF), vascular endothelial growth factor (VEGF), brain derived nervegrowth factor (BDNGF), insulin-like nerve growth factor (INGF), glialfibrillary acidic protein (GFAP), laminin B2, cilliary nerve growthfactor. In addition, anti-inflammatory agents can be used in the methodsof the invention. Examples of such agents include steroids (e.g., DHEA),Kenalog and dexomethason.

The methods of the invention can also comprise contacting the epineuralsheath or nerve graft with, or administering to the individual, one ormore immunosuppressants, particularly in embodiments in which theepineural sheath is of non-autologous origin. Examples ofimmunosuppressants or immunosuppressant protocols which can be usedinclude different types of antibodies (e.g., Thymoglobulin, Camptah,Daclizumab, etc.) and alpha/beta TCR/CsA protocol, Cyclosporine Aprotocol, Tacrolimus, Sirolimus, Rapamycin, Celcept, mycofolenatemoefetil, and/or steroids.

In the methods described herein, it will be apparent to one of skill inthe art that the factors and/or immunosuppressants can be used prior to,at the time of, or after introduction of the nerve graft into theindividual. See, for example, Scharpf, J., et al., Microsurgery,26:599-607 (2006).

As noted above, the nerve graft produced is maintained under conditionsin which nerve tissue is regenerated and/or nerve conduction (partial orcomplete) between the proximal nerve stump and the distal nerve stump isregained. A variety of methods for determining whether nerveregeneration has occurred are provided herein and these as well as othersuch methods are known to those of skill in the art. For example,clinical assessments such as a pin-prick (Siemionow, M, et al., Ann.Plast. Surg., 48:281-285 (2002)) test, electrophysiological tests (e.g.,somatosensory evoked potential evaluation (SSEP)), histomorphometricevaluations, nerve morphometry and morphology evaluations, Tinel sign,nerve conduction velocity, electromyography (EMG), muscle strengthevaluation, etc. can be used (Surgery of the Peripheral Nerve, Susan E.MacKinnon and A. Lee Dellon, Thieme Medical Publishers, Inc., New York,1988).

The epineural sheath of autologous, allogenic, xenogenic or isogenicorigin can be harvested in the form of a full sheath, sheath/strips,and/or conduit and applied, for example, as an epineural sheath graft tofill nerve defects; as an epineural sheath patch to cover nerves andneural tissues protect them from scarring after surgery; as an epineuralconduit to provide nerve guidance at long gap distances or for coverageof spinal nerves; as dura (e.g., for dura tear) of the spinal cord aswell as a patch for coverage of dural and brain defects; and as acombination of these applications (e.g., as a conduit, tube, and patch)for different types of applications in peripheral nerve surgery, plasticsurgery, orthopedics, vascular surgery, spine and neurosurgery.

The epineural sheath is of neural tissue origin, and thus, enhancesnerve regeneration compared to other artificial nerve grafts, conduits,patches. The epineural sheath creates less foreign body reaction and isa natural conduit for the tissues of neural origin. As discussed herein,epineural sheaths can be obtained from a variety of sources (e.g., fromallogenic sources) and can be cold stored (e.g., cryopreserved) for offshelf application. As also shown herein, methods of using epineuralsheaths for treating nerve injuries can be combined with bone marrowstromal cells (e.g., of autologous and/or allogenic origin) forenhancement of nerve regeneration. The epineural sheath described hereincan be used as a graft, conduit, or patch in, for example, peripheralnerve surgery, spine surgery and neurosurgery.

The epineural sheath is of neural tissue origin which provides nerveregeneration, and likely provides less scarring and foreign bodyformation when compared to artificial conduits. The epineural sheathlikely enhances expression of nerve growth factors, neurotrophic factorsand neurotropic factors to promote nerve regeneration and neural tissuehealing in the natural microenvironment. As shown herein, the epineuralsheath works well in combination with bone marrow stromal cells toenhance nerve regeneration which is particularly important in long nervedefects.

The epineural sheath of the invention is an isolated, naturallyoccurring, epineural sheath which, e.g., in its form as a tube and/or apatch, does not include vascular components of the sheath such as vasanervorum, so it is a non-vascularized sheath. It does not includecomponents of tissue response to inflammations such as chemokines,cytokines, or macrophages. In addition, the epineural sheath can befilled with different types of cells including bone marrow derived cellssuch as bone marrow stromal cells, progenitor cells, mesenchymal stemcells, Schwann cells, fat adipocyte cells, chimeric cells, as well as acombination of nerve growth factors, vascular endothelial growth factor,and anti-inflammatory agents such as steroids like DHEA. In addition,the tube may be filled with pulverized epineural sheath, to enhanceregeneration.

Example 1 Repair of Peripheral Nerve Defects with Epineural Sheath Graft

In this study, the potential of using detubulized flat epineural sheathstrip for bridging nerve gaps as an alternative to nerve autograftingtechnique was investigated. Nerve gaps were created by removing a 1.2 cmsegment of the right sciatic nerves. The epineurium of the removedsegment was incised longitudinally and by removing the fascicules, aflat rectangular shaped epineural sheath was obtained. The groups (6rats of each) repaired with one strip, two-strip and full epineuralsheath grafts were compared with the animals repaired with autograftsand the untreated groups at 12th week. Toe-spread was better in ratsrepaired with full sheath grafts and conventional nerve grafts comparedto single strip graft at 12th week. Somatosensory evoked potentialevaluation demonstrated no significant difference in latencies betweenconventional and epineural sheath groups. Histomorphometric results ofthe sections from the animals repaired with autograft, two-strip andfull sheath indicated adequate regeneration. This new technique canserve as an alternative to conventional nerve autografting.

Epineural sheath was previously reported for nerve repair using slidingtechnique, the sleeve technique and recently by the application of theturnover technique, wherein the sleeve has one open end and the oppositeend is a continuation of the damaged nerve and both ends are filled withneural structures such as fascicles, etc. This method is used inexperimental studies and applies only for the autorepair of transectednerves since the epineural sleeve is an integral part of the repairednerve and cannot be moved into a different location. In addition, itapplies only to repair of nerves with a short gap. It does not apply tolarger defects where polyfascicular nerve repair is required and cannotbe used as a free sleeve or tube independent from the damaged nerve.(Atabay, K., et al., Plast. Surg. Forum, 18:121 (1995); Siemionow, M.,et al., Ann. Plast. Surg., 48:281-285 (2002); Tetik, C., et al., Ann.Plast. Surg., 49:397-403 (2002); Ayhan, S., et al., J. Reconstr.Microsurg., 16:371-378 (2000); Yavuzer, R., et al., Ann Plast. Surg.,48:392-400 (2002); Scharpf, J., et al., Laryngoscope, 113:95-101 (2003;Meirer, R., et al., Ann. Plast. Surg., 49:96-103 (2002); Meirer, R., etal., J. Reconstr. Microsurg., 17:625-630 (2001); Demirkan, F., et al.,Ann. Plast. Surg., 34:67-72 (1995)).

As shown herein, a new straightforward method of nerve gap repair usingdetubulized epineural sheath graft for bridging nerve gaps of 12 mm isintroduced. This method is an alternative to nerve autograft techniquewith the advantage of decreased donor site morbidity.

Materials and Methods Animals

Thirty male 8-10 weeks of inbred Lewis rats (LEW) weighing between200-225 g (obtained from Harlan Spraque-Dawley, Indianapolis, Ind.) wereused in the study. All animals received human care in compliance withthe Guide for the care and use of laboratory animals published by theNational Institute of Health. The rats were anesthetized byintraperitoneal pentobarbital (40 mg/kg) administration. All surgicalprocedures were performed by the same surgeon using the samemicrosurgical technique under operating microscope magnification (ZeissOP-MI 6 SD, Carl Zeiss, Goettingen, Germany). Following surgery, animalswere caged individually and were maintained in an environment with a12-hour light/dark cycle. Water and standard laboratory food wasprovided ad libitum.

Surgical Technique

The right sciatic nerves were exposed by an oblique gluteal incision andmuscle splitting technique from the sciatic notch distal to thebifurcation of the tibial and peroneal branches. The left sides were notoperated and were used as controls. The nerve was transected just distalto sciatic notch and proximal to its bifurcation, and 1.2 cm segment ofthe sciatic nerve was removed (FIGS. 1A-1F and 2A-2E). The epineurium ofthe removed nerve segment was incised longitudinally and the fasciculesof the nerve were removed and a flat rectangular shaped epineural sheathgraft was created.

Experimental Groups

Group 1 (n=6)After resection of the 1.2 cm segment of the sciatic nerve, the defectwas left without repair. This group served as a control.Group 2 (n=6)The removed sciatic nerve segment was sutured back using standardepineural technique with 4 interrupted 10/0 nylon sutures placed 90degrees apart.Group 3 (n=6)The flat rectangular epineurial sheath graft was splitted longitudinallyinto two epineural strips and one strip was used to bridge the nervedefect. The single epineural strip was secured to the proximal anddistal stumps of the sciatic nerve by one centrally placed horizontalmattress suture using 10/0 nylon suture (FIG. 3A).Group 4 (n=6)After longitudinal splitting of the epineurium, both epineural stripswere used as a graft to repair the created defect. The strips weresutured to both ends of the sciatic nerve and placed 180 degrees apartfrom each other. Each graft was secured by one epineural 10/0 nylonsuture (FIG. 3B).Group 5 (n=6)The flat rectangular epineural sheath was used to repair the defect as afull graft without longitudinal splitting. The sheath was sutured to theproximal and distal stumps with two epineural sutures placed 180 degreesapart (FIG. 3C).

Following nerve repair in all groups, the gluteal fascia and the skinwas closed with 4/0 vicryl suture. The animals were observed dailyduring the first month follow-up and twice a week thereafter.

Evaluation Techniques Clinical Assessment

The animals were tested by a pin-prick test at 3, 6 and 12 weeks afternerve repair. The pin-prick test was performed by applying pinchingstimulus to the hind-limb skin from the knee to the toes and thewithdrawal of extremity in response to pain stimulus was regarded as apositive pinch reflex (Siemionow, M., et al., Ann. Plast. Surg.,48:281-285 (2002)). The withdrawal reflex at different regions wasgraded from 0 to 3, where the absence of withdrawal was accepted as “0”,withdrawal in response to the stimulus between knee and ankle was gradedas “1”, withdrawal at the proximal plantar surface was graded “2”, andwithdrawal at the toe level was graded “3”.

The toe-spread test was evaluated at postoperative weeks 3, 6 and 12 todetermine motor recovery after repair. The rats were held up by theirtails and their toe-spread movements were graded between 0 and 3. Theabsence of any movement was graded as “0”, the presence of any sign oftoe-spread was accepted as grade “1”, grade “2” indicated presence ofthe abduction of the toes and grade “3” was assigned when both theabduction and extension of the toes was present (Siemionow, M., et al.,Ann. Plast. Sung., 48:281-285 (2002)).

Electrophysiological Test

Electrophysiological assessment was performed by somatosensory evokedpotential evaluation (SSEP) at postoperative weeks 6 and 12 undergeneral anesthesia with intraperitoneal pentobarbital (40 mg/kg). ABio-logic-A-PAC 486 computer (Bio-logic Systems Corp, Chicago, Ill.) wasused for evaluation, which began after the rats were anesthetized withpentobarbital (40 mg/kg intraperitoneal). Stimulating electrodes wereplaced subcutaneously in the operated hind-limb and the ground electrodeto the healthy contralateral hind limb. Using a sagittal incision on thescalp, cranium was exposed by the subperiosteal dissection. Two burrholes were created in the parietal bones of the scull and the recordingelectrodes were placed to the epidural plane over the mid parietalcortex, the active electrode was placed in the contralateral cortex. Thegain of amplifier was set at 3000, with bandpass filter of 30-1500 mHz,stimulation rate of 2.7 cycles/s, stimulus of 100 ms duration, stimulusintensity of 4 to 6 mA, and sweep of 10 msec/div. After 300 trials theaverages were obtained. Testing was then repeated on the other side. Thepositive and the negative potentials of the waveform morphology in theSSEP measurement were obtained and the initial negative wave was markedas N1 and the following positive wave as P1. The second negativepotential was marked as N2. The P1 and N2 potentials were most robustand consistent potentials, therefore P1 and N2 potentials were used forcomparison of sensory recovery between the groups.

Histomorphometric Evaluation Evaluation of Gastrocnemius Muscle Atrophy

In each animal, gastrocnemius muscles were harvested from both sides at12 weeks after repair and wet muscle weight measurements were taken.After formalin fixation, sections were taken from the cross section ofthe muscles and routine H&E stained paraffin sections were prepared.Each slide was evaluated by a blinded pathologist for features ofatrophy. Also mean muscle fiber diameter and mean muscle fibercross-sectional area was calculated from each slide using image pro Plussoftware (Silver Spring, Md., Media Cybernetics).

Evaluation of Nerve Morphometry and Morphology

At postoperative week 12, a 1.5 cm segment of sciatic nerve washarvested including host proximal and distal segment and the connectinggraft of the epineural sheath. The harvested nerve was tied to a woodenstick with 9/0 suture at both ends and fixed overnight in buffered 3.7%gluteraldehyde. A 1.5 cm segment of normal sciatic nerve was harvestedfrom the contralateral side and served as the normal control. Sectionswere taken from the proximal nerve segment, middle segment of the graftand distal segment of the sciatic nerve and were processed in epoxyresin (Spur). Semi thin sections (1 mm) were cut and stained with 1%Toludine Blue.

Sections of normal epineural sheath were also taken before grafting as acontrol for comparison with the grafted sheaths. Both the semi-thin andconventional paraffin sections were prepared to document normalepineural sheath morphology before engraftment.

Photomicrographs of six representative fields were chosen at ×1000magnification from each slide by a blinded observer. After imagecapture, grey scale conversion and contrast enhancement, and automaticcounting were performed using Image pro plus version 6 (MediaCybernetics: MD). The system was calibrated using a 1 mm graticule(micrometer). The following parameters were evaluated; 1-nervecross-sectional area (square mm), 2-myelinated nerve density (per mmsquare), 3-total number of myelinated nerve fibers, 4-axonal diameter(mm) and 5-myelin thickness (mm). A blinded pathologist evaluated eachslide for the maintenance of fascicular architecture, presence ofmacrophages or any other inflammatory cells, presence of nerve debris,fibrosis, neuroma formation, and presence of the foreign body reaction(sutures).

Statistical Analysis

The data was statistically analyzed using Kruskal-Wallis Test forcomparison between groups and Mann-Whitney U Test for comparison withingroups using SPSS 10.0 for Windows. A two sided p value of less than0.05 was considered to be statistically significant. The results areexpressed as Mean+/−SD.

Results Functional Evaluation Pin-Prick Test

In all groups, at 3 weeks after repair, none of the animals responded tothe stimulus at plantar surface. At 6 weeks the animals in Group 2(conventional autografting), Group 4 (two-strip graft) and Group 5 (fullsheath graft) showed better recovery when compared to Group 3 (one-stripgraft) and showed withdrawal at proximal plantar surface. Withdrawal atthe toe level was observed in all groups at 12 postoperative weeks(Table 1). The unoperated animals (Group 1) did not show any sign offunctional recovery.

Toe-Spread Test

None of the groups exhibited toe spread at 3 weeks after repair. At 6thweek, the animals treated with full epineurial grafts (Group 5) andautografts (Group 2) showed better motor recovery demonstratingabduction of the toes. At 12th week, the animals in Groups 2(autograft), and 5 (full sheath graft) showed significantly bettervalues for both the abduction and extension of the toes, indicating fullregeneration, however, the animals treated with one epineural stripdemonstrated only slight movement of the foot without signs of abductionor extension (p<0.01) (FIG. 4) (Table 1). No sign of movement wereobserved in the non-treated group at 12 weeks.

Somatosensory Evoked Potential

The P1 and N2 latency values by SSEP tests are summarized in Table 1.The SSEP evaluations demonstrated non-diagnostic waves at 6th and 12thweek postoperatively in the non-treated group. At 6 weekspostoperatively, the P1 and N2 latencies were significantly prolonged inoperated limbs in treated groups compared to the contralateral normallimbs. These values were comparable with animals treated with the nerveautograft, full epineural sheath grafts and two-strip grafts (FIG. 5).The latencies did not show improvement in the animals treated with onestrip graft, however, there were no statistically significant differencein the P1 and N2 latency values between the treated groups at 12th week.

Histomorphometric Evaluation Gastrocnemius Muscle Evaluation

Macroscopically, the gastrocnemius muscle in each group showed atrophyof the muscle fibers (FIGS. 6A-6C). This was reflected by the lower meanfiber diameter and mean fiber surface area values in all three groupscompared to the normal control. The extent of the atrophy was moreevident in animals treated with the one-strip epineural graft comparedto the other treatment groups (p<0.05).

Nerve Histomorphometry

Macroscopic observations of the unrepaired rats showed that there wasextensive fibrosis between the nerve stumps without visible neuralarchitecture. In the conventional autograft group, the two-strip graftgroup and the full sheath graft group there was a normal nervestructure, however the nerves were smaller in diameter compared to thenormal control (FIGS. 7A-7C and 8A-8B). The sections taken from the hostproximal segment and the contra-lateral normal nerve showed comparablediameter and density of myelinated nerve fibers. In general, theepineural sheath grafts showed reduced nerve cross-sectional area. Thesections taken from the graft showed presence of clusters of smalleraxons with thin myelin sheath and complementary shape (regenerativeclusters) (FIGS. 9A-9C). The axons did not show any fasciculararchitecture in the graft. There was an absence of inflammatory cells,nerve debris, fibrosis or neuroma formation. The results ofhistomorphometric analysis are summarized in FIG. 10 and Table 3.Sections from the distal segment showed increased nerve cross-sectionalarea compared to the epineural sheath graft. There was a presence of thefascicular architecture as well as presence of the extra fascicularregenerative axon clusters. The myelinated axon count in the fullepineural sheath grafts and in the two-strip epineural sheath graftsshowed a significant increase in the axon count compared to the proximalsciatic nerve segment, which showed typical regenerative response whereaxons were replaced by the regenerative axon clusters. However, in fourout of six animals treated with one-strip epineural graft there was aminimal nerve regeneration presented by a very low numbers of myelinatedaxon and small diameters of myelinated axons. The other two animals fromthis group showed adequate nerve regeneration. Nerve cross-sectionalarea, total number of the myelinated fibers, and the axon diameter weresignificantly increased in the rats treated with autograft and fullsheath grafts compared to the rats treated with one-strip epineuralsheath graft (p<0.005). Analysis of the histomorphometric parameters,such as the nerve cross-sectional area, total number of the myelinatedfibers, the axon diameter, fiber diameter and fiber cross-sectional areadid not show significant difference between the full epineural sheathgraft group and the autograft technique groups. Myelinated fiber densitywas found to be higher in the rats treated with full sheath graftcompared to the rats repaired with the autograft (p<0.005). Myelinthickness was significantly higher in the autograft group compared toother groups (p<0.05).

Sections taken from epineural sheath before engraftment showed presenceof the fibrocollagenous tissue with the surrounding blood vessels. NoSchwann cell was identified histologically.

Discussion

Nerve regeneration is a complex phenomenon involving interaction betweenthe Schwann cells, axons and other cells such as recruited macrophages(Hall, S., Hand Surg. [Br], 26:129-136 (2001)). Following injury, thedistal axon and its myelin sheath degenerates and the debris are removedby the recruited macrophages. The Schwann cells de-differentiate leadingto the down regulation of the expression of the myelin related m-RNAsand up regulation of the expression of receptors related to the axonallyderived ligands and neurotrophins (Scherer, S S, Peripheral neuropathiesin “The Molecular and Genetic Basis of Neurologic and PsychiatricDisease”. Butterworth-Heinemann; 435-439 (1993)). This phenotype switchof the Schwann cells facilitates axon in-growth and extension. Theproliferating and de-differentiating Schwann cells line up within eachbasal lamina tube to form the bands of Bunger. Meanwhile, a growth coneforms at the viable tip of the distal axon from which multiple axonsprouts emerge in order to occupy the place previously taken by a singlelarge myelinated axon. Such formations are called “regenerative cluster”and are defined in the presence of three or more closely situatedmyelinated axons. Interaction with the in-growing axons causes a changein the Schwann cell phenotype again, which reverts back to the myelinproducing form. Eventually sufficient number of the regenerating axonsreaches appropriate target organs for the restoration of function.

In the presence of nerve defect secondary to the peripheral nerveinjury, this gap is filled with an exudate that contains bloodcorpuscles and macrophages forming a fibrin clot. The fluid in the gaphas many soluble factors that have high neurotrophic activity of factorssecreted by the divided peripheral nerve stumps, which accelerates nerveregeneration (Hudson, T W., et al., Clin. Plast. Surg., 26:485-497(1999)). During the first few days a fibrin forms a longitudinallyoriented matrix bridging the two stumps, in the second week there is aningrowth of the capillaries, fibroblasts and Schwann cell into the gapzone from both nerve stumps (Hudson, T W., et al., Clin. Plast. Surg.,26:485-497 (1999)). Regenerating axons pass through this matrix andreach the distal stump followed by myelinization. Lack of structuresbetween the nerve ends to guide regenerating axons may causemisdirection of the axons and neuroma formation. It was stated thatencasing the stumps of a transected nerve by a tube allows accumulationof locally produced neurotrophic factors (Hazari, A., et al., Br. J.Plast. Surg, 52:653-657 (1999)). Lundborg have demonstrated that in theabsence of a conduit, regenerating axons fail to reach the distal stumpin the presence of 10 mm nerve defect. However, regeneration can occurin 15 mm defect when a silicon chamber is used to bridge the defect(Lundborg, G., et at, Exp. Neurol., 76:361-375 (1982)). If the distalend is left open, regeneration is only partial confirming importance ofthe distal stump. The target muscles are also important in nerveregenerating as the nerve does not regenerate when the distal segment,which is extending to the corresponding muscle, is resected. Thisconfirms the importance of muscle-nerve interactions. Seckel et al. haveshown the importance of the distal nerve stump as a source of theneurotrophic factors by demonstrating failure of regeneration across thegap in the absence of the distal stump or at a distance greater than 10mm from the proximal stump (Seckel, B R., et al., Plast. Reconstr.Surg., 74:173-181 (1984)). By using Y-configured chambers, MacKinnon etal. have shown that regenerating axons selectively have grown down tothe channel of the tube that contained distal nerve stump (Mackinnon, SE, et al., J Hand Surg. [Am], 11:888-894 (1986)). Therefore for therepair of peripheral nerve defects, conduits in the form of thetubulized chambers were used in an attempt to prevent multidirectionalaxonal sprouting and to help the axons to reach the distal target.

Many different strategies have been developed over the years tofacilitate nerve regeneration following injury to peripheral nerve(Siemionow, M., et al., Neurol. Res., 26:218-225 (2004)). Restoration ofthe peripheral nerves following nerve injury by application of standardnerve grafting technique is limited due to the limited availability ofthe donor site and the morbidity. Different type of nerve grafts havebeen used including application of nerve allografts underimmunosuppression protocols as well as use of the acellular nerveallografts (Siemionow, M., et al., Ann. Plast. Surg., 24:63-73 (1997);Scharpf, J., et al., Laryngscope, 113:95-101 (2003); Meirer, R., et al.,Ann. Plast. Surg., 49:96-103 (2002); Doolabh, V B., et al., Plast.Reconstr. Surg., 103:1928-1936 (1999); Kim, B S, et al., J. Biomed,Mater. Res., 68A:201-209 (2004)). The adverse effects of theimmunosuppression prevent routine clinical application of the allografttechnique in the clinical practice. Significant delay observed duringregeneration when using acellular nerve allograft makes this techniquenot clinically applicable. Other investigations of new proceduresfacilitating nerve regeneration range from the administration ofhyperbaric oxygen and Acetyl-1-carnitine to the use of various types ofnatural and bioengineered conduits with or without impregnation withnerve growth factors (Zamboni, W A, et al., J. Reconstr. Microsurg.,11:27-29 (1995); McKay Hart, A., et al., Neurosci. Lett., 334:181-185(2002); Kelleher, M O, et al., Br. J. plast. Surg., 54:53-57 (2001);Midha, R., et al., J. Neurosurg., 99:555-565 (2003)). Described hereinis conduit material that causes minimal inflammatory reaction, andserves as a structural guide for regenerating axons and stimulatesaxonal regeneration along its entire length.

As described herein, whether the bridging of a short nerve gap with theepineural sheath graft is sufficient to sustain nerve regeneration wasstudied. If the answer is positive, then the question arised if the sametechnique of epineural sheath grafting process will be adequate enoughto be applied to bridge longer nerve gaps. Epineurium was previouslyused for peripheral nerve repairs in the experimental models in rats(Atabay, K., et al., Plast. Surg. Forum, 18:121 (1995); Siemionow, M.,et al., Ann. Plast. Surg., 48:281-285 (2002); Tetik, C., et al., Ann.Plast. Surg., 49:397-403 (2002); Ayhan, S., et al., J. Reconstr.Microsurg., 16:371-378 (2000); Yavuzer, R., et al., Ann Plast. Surg.,48:392-400 (2002)). The cooptation side was wrapped by a slidingepineural sheath tube or turnover epineural sheath tube and it wasdemonstrated that by using this technique there was a less inflammatoryreaction with less fibrosis at the cooptation site (Yavuzer, R., et al.,Ann Plast. Surg., 48:392-400 (2002)). Epineural sleeve technique, whichwas first introduced to prevent neuroma formation and facilitate nerveregeneration, was compared with the conventional nerve repair and betterfunctional and histomorphometric results were reported (Siemionow, M.,et al., Ann. Plast. Surg., 48:281-285 (2002); Tetik, C., et al., Ann.Plast. Surg., 49:397-403 (2002)). However these experimental models ofepineurial sheath application were used in the tubulized form for theprimary repair of the transacted nerve without a large gap. In thepresence of a nerve gap, these sliding or turnover epineural sheath tubemodels cannot be applied as there will be lack of the nerve segment ofthe sufficient length to dissect the epineural tube of the distal orproximal nerve stump. It is also impossible to dissect the tube from adistant donor nerve without impairing the nerve's continuity. Finally,the sliding or turnover tubes are always an integral part of therepaired nerve and as such contain normal nerve fascicles inside thetube and cannot be removed from the nerve for distant applications,preservation or storage. Described herein is an epineural sheathgrafting model applied for repair of nerve defects. Using a flat fullepineural sheath graft, the functional, electrophysiological andhistomorphometric outcomes comparable to the conventional repair withthe nerve autograft were achieved. Flat rectangular shaped epineuralsheath can easily be obtained without impairing nerve continuity. It wasshown that harvesting of the epineurium does not alter function of thedonor nerve and the donor site is covered by the remaining epineuriumafter a period required for healing (Yavuzer, R., et al., Ann Plast.Surg., 48:392-400 (2002)). Therefore, this method brings the advantageof eliminating the donor site morbidity such as anesthesia, sensoryimpairment and neuroma formation, which are the major drawbacks whenautografts are used.

As shown herein, detubulized epineural sheath graft provided similarfunctional recovery and histomorphometric findings compared to theconventional nerve repair with the autograft. The epineural sheathgrafts described herein can be used for repair of nerve defects as analternative method to the autograft technique without donor sitemorbidity.

TABLE 1 Pin-prick test, toe-spread test, and SSEP evaluation results atpostoperative 12^(th) week. SSEP (milliseconds (ms)) Non-Operated siteOperated Site Groups Pin-prick Toe-spread P1 N2 P1 N2 Autograft 3.0 ±0   2.83 ± 0.40 16.38 ± 0.89 21.56 ± 1.14 17.47 ± 1.62 24.84 ± 2.14One-Strip 2.33 ± 0.51 1.16 ± 0.40 15.79 ± 0.67 22.61 ± 3.00 19.03 ± 3.0226.25 ± 2.48 Two-Strip 2.83 ± 0.40  2.0 ± 0.63 16.19 ± 1.24 22.58 ± 3.1017.47 ± 1.57 24.98 ± 2.08 Full 2.66 ± 0.51 2.16 ± 0.40 16.19 ± 0.7822.47 ± 0.84 16.24 ± 1.85 24.61 ± 2.36 Sheath

TABLE 2 The ratio of wet weight of gastrocnemius muscle in operated siteto non operated control side at postoperative 12 weeks. Groups WO/WCConventional 0.48 ± 0.10 One-Strip 0.23 ± 0.13 Two-Strip 0.38 ± 0.09Full Sheath 0.47 ± 0.09 WO: Wet weight of the gastrocnemius muscle inthe operated side WC: Wet weight of the gastrocnemius muscle in thenon-operated contralateral side

TABLE 3 Histomorphometric results in five experimental groups. NerveMyelinated Fiber Cross- Fiber Total Axon Myelin Fiber Cross- SectionalDensity Myelinated Thickness Thickness Diameter Sectional Groups area(mm²) (mm²) Fiber (μm) (μm) (μm) Area (μm) Control 0.41 ± 0.06 18936 ±1520 7901 ± 1450 5.05 ± 0.16  2.04 ± 0.07 34.8 ± 0.70 965.6 ± 63.5 SideConventional 0.16 ± 0.02 25690 ± 773  4299 ± 687  2.7 ± 0.07 0.96 ± 0.1013.0 ± 0.28 131.8 ± 6.94 One- 0.10 ± 0.01  16510 ± 12049 1798 ± 1453 1.5± 0.48 0.60 ± 0.14 11.6 ± 1.44  99.3 ± 28.3 Strip Two- 0.15 ± 0.02 25018± 3669 3868 ± 609  2.7 ± 0.53 0.78 ± 0.13 12.96 ± 0.46  130.5 ± 6.6 Strip Full 0.16 ± 0.03 33550 ± 3472 5596 ± 1357 2.85 ± 0.51  0.70 ± 0.1213.1 ± 0.39 131.8 ± 12.9 Sheath

Example 2 Enhancement of Neural Regeneration of Peripheral Nerve Defectsby Epineural Tube Graft Enriched with Donor Derived Bone Marrow StromalCells

Nerve grafting has been the most widely accepted surgical interventionfor the repair of peripheral nerve defects. Traditionally however, suchinterventions have been associated with varying degrees of donor sitemorbidity. In the search for alternative methods of peripheral nerverepair, the use of biological and artificial materials for the creationof nerve conduits has been investigated. This study described herein wasperformed to assess the effects of bone marrow stromal cells (BMSC) innerve gaps repaired with isogenic, isolated epineural tubes filled withisogenic and allogenic BMSCs.

Methods

A total of 54 isolated epineural tubes were harvested. Access to theperipheral nerve e.g., sciatic nerve was made by skin incision andsubcutaneous tissue dissection down to the anatomical location of thenerve. At this level the sciatic nerve was cleared of all surroundingtissues by blunt dissection as far proximally as the sacral plexus andas far distally as its division into the terminal nerve branches. Allcollateral branches arising from the sciatic nerve throughout its lengthcan be detached and used separately to create an epineurial sheathtubular grafts of different size diameters and lengths. At this pointthe sciatic nerve was ready to be dissected out. The nerve wastransected as proximal as was feasible at its origin from the sacralplexus, and then transected distally where the nerve divides into itsterminal components, at the level of insertion into the muscle.Depending on the area of nerve harvest the nerve was then suspended/on aeither straight driver/irrigator with round tip (e.g. 30 gauge.times.25mm depending on nerve diameter—the driver diameter must be smaller thannerve diameter) or on the curved/hook finished driver/irrigator or onscrewdriver type of irrigator. The irrigator was filled with chilledsolution (either cryopreservation solution for long term storage, ornerve culture medium or combination of both—depending on the fate ofgraft) and was kept moist on the dissection board by soaking it with0.9% Sodium Chloride.

Under microscope or loop magnification the axons were then gently teasedfrom its epineural sheath with the use of circular motion ofdriver/irrigator and jeweler fine forceps pulling the sheath away fromthe axons and driver in the “devaginating” maneuver—so the axon fiberswere pulled from the distal end whilst the epineural sheath was heldfrom the proximal end on the driver/irrigator. Once all axons includingperineurium and endoneurium were removed the intact, clear epineuralsheath was left as a product of this process and was then inspected forintegrity. Two different types of epineural tubes can be made sensorytubes and motor tubes based on the type of nerves from which they wereharvested. Different lengths and diameters of epineural sheaths may beobtained by this process based on the nerve diameters.

The epineural tubes were transplanted in 3 experimental groups (18animals in each group). Group 1 was control saline, Group 2 isogenic(Lew RT1.sup.1) BMSCs and Group 3 allogenic (ACI RT1.sup.a) BMSCs. Theeffect of BMSCs and nerve growth factor in epineural conduits wasevaluated in a rat sciatic segmental defect model (FIG. 12). In Groups 2and 3, BMSCs were stained with PKH-26 dye before transplantation, toassess BMSCs nerve engraftment and migration. After staining2.5-3.0.times.10.sup.6 cells were delivered directly into thetransplanted epineural tube. Evaluations were performed at 6, 12 and 18weeks post-transplant. Sensory and motor recoveries were evaluated byGastrocnemius Muscle Index (GMI) pinprick, toe-spread and Somato-SensoryEvoked Potentials (SSEP). Axonal counting was performed in addition toimmunostaining following nerve growth factors: nerve growth factor(NGF), Laminin B2, glial fibrillary acidic protein (GFAP), and vascularendothelial factor (VEGF) and Von Willebrand Factor for the assessmentof the expression of neurotrophic factors and regenerative potential oftransplanted BMSCs.

Results

6 weeks post transplantation all groups scored 3 on the pin-prick test.Toe spread for groups 1, 2 and 3 were respectively 1.7; 2; 1. SSEPs ingroups 1, 2 and 3 (P1, N2-latencies (milliseconds); P1, N2 percentages(%) of normal values) were respectively (20.2; 23.6; 113; 95), (17.5;18.1; 98; 73) and (15.7; 21.65; 88; 87). GMIs in groups 1, 2 and 3 wererespectively (0.45; 0.48; 0.47). Group 2 showed a higher number ofregenerated axons (90.6.±0.26.9) compared to Group 1 (71.4.±0.3.0) and 3(76.4.±0.5.4).

Immunostaining with NGF and laminin B2 assessed the expression ofneurotrophic factors and the regenerative potential of transplantedBMSCs within the epineural conduits. Histology showed the first signs ofaxonal regeneration in all groups at 6 weeks. The two groups with BMSCshad PKH26 positive cells in the proximal part of the transplantedepineural conduit. Immunostaining with NGF confirmed upregulation of NGFin the proximal segments of the conduit compared to the middle anddistal parts. Differentiation efficacy was greater after transplantationof isogenic BMSCs compared to allogenic BMSCs. NGF upregulation in theMSC-containing groups correlated with upregulation of laminin B2 in bothgroups, indicating active nerve regeneration. Better functional recoveryand axonal regeneration was seen in those epineural conduits thatcontained BMSCs compared to those that contained only saline. FIG. 12shows the BMSC processing steps from harvest to implantation, and FIG.13 shows the histology results for nerve regeneration for the threegroups whereas FIG. 14 shows immunostaining for NFG and BMSC andpresence of coexpression of NGF/BMSC at 6 weeks at proximal segment ofthe tube. FIG. 14A shows immunostaining for NFG and BMSC and presence ofcoexpression of NGF/BMSC at 6 weeks at proximal segment of the tube.

In groups 2 and 3 (with BMSCs) PKH positive cells in addition to NGF,Laminin B2, GFAP, VEGF and Von Willebrand Factor were found in theproximal segment of the transplanted tubes. In contrast, at 18 weekspost transplant isogenic stromal cells (Group 2) expressed neurotrophicfactors in the distal portion of the transplanted nerve (see FIG. 14B).This confirmed advancement of nerve growth/regeneration over the entire(previously empty) tube segment over an 18 week period. In the allogenicgroup (Group 3) neurotrophic factors were expressed in the middleportion of the transplanted nerve indicating slower but advancinggrowth/regeneration. Coexpression of NGF-staining in combination withPKH-staining confirmed that BMSCs differentiated into neural tissues.Upregulation of Laminin-B2 in both groups indicated active nerveregeneration. Finally, Group 1, the saline control group, showednegative staining for NGF indicating that there is no “spontaneous”nerve regeneration and that BMSC are essential for nerve growth over theempty tube segment.

Conclusion

Co-transplantation of BMSCs within epineural tubes resulted in nervegrowth/regeneration over the peripheral nerve defects and confirmed theregenerative potential of BMSCs through their differentiation into andcreation of neural tissue. Isogenic BMSCs showed a greater regenerationpotential to create neural tissue than allogenic BMSCs. These findingscorrelated with functional recovery of the nerves as measured by SSEPand axonal regeneration utilizing electron microscopy.

Example 3 Immunostaining of the Epineural Tube Methods:

The sciatic nerves were harvested from naive Lewis rats (Lew RT1.sup.1).The fascicles were removed using fascicle-stripper device. Afterfascicle removal empty epineural tube was created. This tube was nextevaluated for expression of nerve factors involved in nerve growth andregeneration and was evaluated by monoclonal antibodies andimmunofluorescence technique.

Next the cross-sections and longitudinal sections were prepared fromwhole tubes as well as from tubes split opened and let flat for thetesting.

These freshly dissected nerve tubes from naive Lewis rat weresnap-frozen in liquid nitrogen. Prior to immunostaining tissue slideswere cut for 5 .mu.m slides and fixed for 10 min. in acetone. Next,sections were rinsed in TBS (Dako) buffer and incubated with mouseanti-rat NGF (H-20), GFAP (2E1), VEGF (C1) (Santa Cruz Biotechnology,Inc), S100 (clone 4C4.9) (LabVision), MHC class II (clone OX-17) andLaminin B (clone D18-2.2) (BD Pharmingen, CA) monoclonal antibodies for30 min. The binding of primary antibodies was detected using a rabbitanti-mouse immunoglobulin/FITC (DAKO, Carpinteria, Calif., USA) inaccordance with the manufacturer's instructions. Slides were mounted inVectashield mounting medium with DAPI for fluorescence and analyzedusing fluorescence microscope.

Results

Only Laminin B was constitutively expressed on the epineurium of thenaive Lewis rats. There was also weak expression of S-100, GFAP andVEGF. In contrast, NGF, MHC class II and VWF were not detected infreshly isolated epineural tubes of naive rats.

TABLE 4 Cross-Section Longitudinal- Longitudinal- Antibody EpineuralTube Section Tube Section Flat Tube NGF neg neg neg Laminin + ++ ++ S100w+ w+ w+ GFAP w+ w+ w+ VEGF w+ w+ w+ vWF neg neg neg MHC class II negneg neg w+—weak expression

Immunostaining results showing laminin B expression in the epineuraltube are shown in FIG. 16.

Example 4 the Effects of Inflammation on GFAP Expression in SatelliteCells of the Dorsal Root Ganglia

Damage to the peripheral nervous system (e.g., peripheral nerves, dorsalroot ganglia, and dorsal roots) or the central nervous system (CNS)(e.g., i spinal cord, thalamus) often results in neuropathic pain.Compression of the spinal cord and nerve roots can result inirreversible histological and physiological changes such as intraneuralfibrosis, demyelination, and neuronal loss. The mechanism behind thesechanges is not clearly understood however, it is likely that satellitecells (SC) play a key role. Satellite cells are believed to beneurological cells that closely intact with nerve cells of the dorsalroot ganglion (DRG). Satellite cells are connected by gap junctionswhich are involved in the spatial buffering of extracellular K+ and inneuroprotection.

GFAP expression increases in response to CNS injury, neurodegenerativedisease (e.g., Alzheimer's dementia) and aging. Increased expressioncorresponds to a characteristic cellular hypertrophy referred to asastrogliosis. In the CNS, GFAP plays a key role in modulating astrocyticand neuronal glutamate transporter trafficking and function. Activationof glutamate receptors on astrocytes leads to an increase inintracellular Ca2+. Increased Ca2+ levels results in activation ofsynaptic receptors and alteration in synaptic transmission.

GFAP is also responsible for maintenance and vascular permeability atthe blood-tissue interface. Loss of GFAP impairs Schwann cellproliferation and delays nerve regeneration after injury. GFAP is alsoessential for normal white matter architecture and blood-brain barrierintegrity, and its absence leads to late-onset CNS dysmyelination.

Astrocytes synthesize and release factors, such as colony-stimulatingFactor (CSF) or chemokines (e.g., CCR2 ligand) which induces microglialactivation. Microglia can synthesize and release IL-1b, IL-6 andTGF-.beta. which play a role in pain sensitization, presumably via aspinal mechanism.

The effects of inflammation on the expression of GFAP in satellite cellsof the dorsal root ganglion were tested. Satellite cells are involved inthe pathogenesis of neuropathic pain.

Methods/Materials

lumbar radiculopathy model (Kawakami, M., et al., Spine,19(16):1780-1794 (Aug. 15, 1994))

96 adult Lewis rats underwent a hemilaminectomy; the contralateral sidewas not exposed

cat gut suture was place topically on the exposed DRG

animals were then sacrificed at 6 hrs, 24 hrs, 48 hrs, 72 hrs and 7 dayspost operatively and their DRG harvested

all NC and SC were counted

GFAP expression was assessed by counting all SC that express GFAP anddividing them by the total number of SC

Results

164 DRGs were harvested and available for analysis

naive controls did not express GFAP

GFAP expression was observed in 30% of SC and SC sheaths at 6 hrs

GFAP expression was observed 85% of SC and SC sheaths at 24 hrs

GFAP expression was observed in 100% 48 hrs, 72 hrs and 7 days SC and SCsheaths

in the contralateral internal controls 5% of SC and SC sheaths expressedGFAP at 6 hrs

20% controls at 24 hrs

30% controls at 48 hrs

100% controls at 72 hrs and 7 days

Discussion

increase in GFAP expression by SC leads to activation of the glutamatereceptor

this leads to microglia activation and cytokine release

this study supports the role of satellite cells in the development andmaintenance of nerve injury-induced neuropathic pain

Conclusion

under physiologic conditions, the expression of GFAP by SC isundetectable by immunohistochemistry

as the inflammation process develops, GFAP expression increases, with30% of SC at 6 hrs and 85% of SC at 24 hrs being GFAP immunoreactive

neither the contralateral internal control nor the sham group displaysuch a profound increase in GFAP immunoreactivity at 6 hrs and 24 hrs.

Example 5 Use of the Epineural Sheath as a Patch

Compression of the spinal cord and nerve roots can result inirreversible histological and physiological changes such as intraneuralfibrosis, demyelination, and neuronal loss. The epineural sheathsdescribed herein can be used as a protective anti-inflammatory sheath inpatients undergoing decompression procedures for myelopathy secondaryto, e.g., sponylosis, disc herniation, trauma, tumor and/orcomplications associated with diabetes.

For example, an epineural sheath of the desired size is placed topicallyon the exposed dura. The sheath uses the surface properties of thesheath-dura interface to adhere to the dura. Alternatively, glue orsuture can be used selectively to enhance epineural sheath adhesion.

The epineural sheaths described herein can also be used to increaseneural regeneration in patients undergoing decompression procedures formyelopathy secondary to e.g., spondylosis, disc herniation, trauma,tumor, and/or complications associated with diabetes.

For example, an epineural sheath of the desired size is placed topicallyon the exposed dura. The sheath uses the surface properties of thesheath-dura interface to adhere to the dura. Alternatively, glue is usedselectively to enhance epineural sheath adhesion.

The epineural sheaths described herein can also be used as a dura matersubstitute in cases of e.g., dural deficit, iatrogenic durotomy, and/ordural transplant

For example, a patch of desired size is cut out of a premade epineuralsheath. This patch is then secured into place using sutures/staple/glue.

The epineural sheaths described herein can also be used to preventscarring and adhesions in patients e.g., undergoing decompressiveprocedures of the spinal cord, thecal sac, and or nerve roots, and/or inpatients suffering from radiculopathy.

The epineural sheath of desired size described herein is placedtopically on the exposed dura. The sheath uses the surface properties ofthe sheath-dura interface to adhere to the dura. Alternatively, gluecould is used selectively to enhance epineural sheath adhesion.

The epineural sheaths described herein can also be used to increaseneuronal regeneration and decrease inflammation in patients withradiculopathy (e.g., injectable form).

The epineural sheath of desired size described herein is placedtopically on the exposed dura. The sheath uses the surface properties ofthe sheath-dura interface to adhere to the dura. Alternatively, glue isused selectively to enhance epineural sheath adhesion.

The epineural sheath described herein can also be used to create anoptimal microenvironment and increase neuronal regeneration in patientssuffering from spinal cord injury.

The epineural sheath of desired size described herein is placedtopically on the exposed dura. The sheath uses the surface properties ofthe sheath-dura interface to adhere to the dura. Alternatively, glue isused selectively to enhance epineural sheath adhesion.

An examples of a protocol for epineural sheath harvesting is describedherein. Patches are created by splitting of the tubular epineural sheathlongitudinally. This is done after removal of the axons. The straightstripper-irrigator is inserted into the tubes and the proximal anddistal edges of the tube is fixed to the harvesting board. Next, usinge.g., a miniscalpel or microscissors, longitudinal transsection isperformed under the guidance of the irrigator. Once transsected, theepineural sheath is opened like a book, flattened, checked for integrityand stored either by cryopreservation technique or cold stored in 4degrees Celsius.

Different lengths and sizes of sheath can be harvested based on nervelength and diameters. Also the patches can be made thicker bymultiplying layers of single patches and can be made in largerdimensions by combining several patches together for differentdimensions e.g. 2.times.2 cm 4.times.4 cm 6.times.6 cm etc.

Two different types of patches can be made such as sensory patches,motor patches and mixed sensory/motor patches based on the type ofnerves from which they were harvested.

Alternatively patches may be created after cryopreservation from theepineural sheath tubes.

Cryopreservation Technique

For long term preservation and storage epineural sheath tubes andpatches can be submitted to cryopreservation techniques.

The goal here is two-fold, first it keeps products stored for as long as10 years or more in the tissue bank or company storing facility.

Second, cryopreservation is reduces immunogenicity of the epineuralsheath but at the same time is keeps the integrity and cellularcomponents of the epinerium intact. An example of a detailedcryopreservation protocol is outlined below.

Cryopreservation Protocol I. Equipment and Supplies Media Preparation

1. Sterile 5 ml serological pipets2. Sterile 10 ml serological pipets3. Culture media flasks4. Sterile 100 ml bags (saline bags)—2+3 pcs5. Sterile nalgene filters 0.2 mm

6. Needle 20 G—2+3 pcs 7. Syringe 20 ml 2+3 pcs 8. Crushed ice

9. Reagents—see point No III.

Epineural Sheath Harvesting as Described Above Prefreezing Perfusion

1. Infusion pump2. Infusion lines 2 pcs3. Culture plate

4. Syringe a 10 ml

5. Long needle (spinal)6. Sterile forceps7. Planer temperature-controlled freezer

8. Cryovials (5 ml) 9. Cryocanes 10. Cryosleeves 11. Cryomarkers 12. LN2Dewar Post Thawing Perfusion

1. Infusion pump groin flap cryopreservation protocol 22. Infusion lines 3 pcs3. Warm bath 37.degree. C.4. Culture plate5. Sterile falcone container

6. Crushed ice Reagents

1. Dimethyl sulphoxide (DMSO)

1.5 M (final concentration)

Mol. Weight: 78.13

Sigma-Aldrich, St. Louis, Mo., Catalog #: 15,493-8

2. Leibovitz L-15 medium

Irvine Scientific, Santa Ana, Calif., Cat #9082

3. Fetal bovine serum (FBS)

10% (final concentration)

Irvine Scientific, Santa Ana, Calif., Cat #3000

4. Sucrose

0.25, 0.1 M (final concentration)

Mol. Weight: 342.3

Sigma-Aldrich, St. Louis, Mo. Cat #S-7903

5. Lactated Ringer's solution: Heparinized (5 U/ml)

II. Preparation of Reagents Use Sterile Technique

To prepare 100 ml of Cryoprotective Media:

Transfer 79.36 ml of Leibovitz L-15 media to a sterile flask.

Add 10 ml of inactivated FBS and mix (55.degree. C. for 30 minutes)

Add 10.64 ml of DMSO and mix

Filter media through Nalgene Filters (0.2 mm)

Place media in the refrigerator until needed

Transfer to sterile empty bag for infusion

Tissue Preparation

Transfer some of the Leibovitz media to another sterile tube fortransport to OR, keep media cold, and provide sterile 150 mm.times.25 mmsterile culture dishes

Flap with a pedicle are harvested, cannulated in Microsurgery lab anddelivered to Cryopreservation lab.

-   -   The tissue is placed in sterile culture dish containing the cold        media    -   The tissues are transported on ice

Infusion System Set-UP

Set-up the infusion line to infusion system

-   -   Connect infusion system into the cannula through the artery    -   Infusion rate: 80 ml/h    -   Limit of infusion pressure: 200 mmHg

Start of Perfusion on the Ice

-   -   Perfuse with Heparinized (5 IU/ml) Ringer's solution (room        temperature) till the fluid comes out from the vein for 10        minutes.        groin flap cryopreservation protocol 3    -   Perfuse with the Cryoprotectant (0.75 M DMSO+10% FBS in        Leibovitz L-15 media) for 15 minutes    -   Perfuse with the Cryoprotectant (1.5 M DMSO+10% FBS in Leibovitz        L-15 media) for 15 minutes

III. Preparation to Cryopreservation 1. Place Tissue to Cryovials

2. Label 5 ml cryovials with tissue #, date, and other information3. Add 1 ml of cold cryoprotectant to each tube4. Transfer one flap to each tube5. Fill up the cryovial with cryoprotectant solution6. Transfer vials to Programmed Planer Freezer

IV. Planer Freezer Set-Up

-   -   Connect the liquid nitrogen hose to the LN2 tank. Turn the knob        and open the LN2 valve.    -   Make sure that the cryochamber is tight and all the four knobs        are in the horizontal position    -   Switch on Planer Freezer

The slow freeze settings are:

-   -   Temperature drop down to 4.degree. C.    -   Cool at −2.degree. C./min to −7.degree. C.    -   Soak for 10 minutes    -   Manual seeding,    -   Continue to cool at −0.3.degree. C./min to −40.degree. C.    -   Cool at a rapid rate of −25.degree. C./min to −140.degree. C.    -   Wait for chamber temperature to drop down to 4.degree. C.

Load specimen vials

V. Long-Term Storage of Issues in the LN2 Tank

-   -   The samples can be stored in LN2 tanks.        cryopreservation protocol 4

VI. Preparation of Reagents for Thawing

Three solutions with different concentration of sucrose are needed

1. 0.25M Sucrose Leibovitz Solution

Transfer 90.0 ml of Leibovitz L-15 media to a sterile flask

Add 0.25 M: 8.56 g of Sucrose

Add 10 ml of FBS in media Filter media through Nalgene Filters (0.2 mm)Transfer to sterile empty bag for infusion

2. 0.1M Sucrose Leibovitz Solution

Transfer 90.0 ml of Leibovitz L-15 media to a sterile flask

Add 0.1M: 3.42 g of Sucrose

Add 10 ml of FBS in mediaFilter media through Nalgene Filters (0.2 mm)Transfer to sterile empty bag for infusion3. Non sucrose Leibovitz solutionTransfer 90.0 ml of Leibovitz L-15 media to a sterile flaskAdd 10 ml of FBS in mediaFilter media through Nalgene Filters (0.2 mm)Transfer to sterile empty bag for infusionPlace all media in the refrigerator until needed, transport on ice.

VII. Thawing the Cryopreserved Tissue

1. Remove the Cryovials from the Dewar2. Hold for 5 minutes in thinsulated container3. Hold for 5 minutes at room temperature4. Plunging and swirling in a water bath at 25/37.degree. C. with gentleshaking for 5 minutes5. Quickly empty the contents of vial into a Petri dish containingLeibovitz L-15 medium with 10% fetal bovine serum6. Assemble the cannula, tubing and infusion pump7. Perfuse the flap at a rate 80 ml/h (max pressure 200 mmHg) for 30/45minutes using 0.25 M, 0.1 M and 0 M Sucrose to remove thecryoprotectant. Perfusion time should be 10/15 minutes for each step.8. Wash with culture media. Place in Non sucrose Leibovitz solution fortransportation

VIII. Transplantation Technique—Optional

1. Keep sheath immersed in Leibovitz solution2. Wash with heparinized ringer's lactate solution (5 U/ml)3. Wash or keep irrigated with neuroprotective (nerve growth factorcontaining) solution

4. Optionally:

a. Give systemic heparineb. Give systemic heparine+aspirinec. GiveNote: Thawing protocol of epineural tissues should be done on ice asdescribed above

Example 6 Application of the Epineural Sheath as a Protective Patch forDRG after Laminectomy at L5 Level

A dorsal midline incision was made and was carried down sharply to thespinous process of an adult Lewis rat. A combination of sharp and bluntdissection was used to dissect the lumbar musculature over the spinousprocess and the lamina of L5. Two retractors were placed to retract thedorsal musculature. A rongeour was used to remove posterior bonyelements on the left side, exposing the dorsal root ganglia (DRG). Thedorsal root ganglia were dissected using microdissectors. The epineuralsheath which was harvested from the sciatic nerve of a donor rat of thesame species (isogenic) was circumferencially wrapped around the dorsalroot ganglia of the L5 on the left side which was exposed. Thecontralateral side was left intact and the fascia was closed with 4-0Vicryl and the skin was closed with 4-0 Vicryl as well.

Seventy two hours after patch application, the animals were sacrificedand their DRG and accompanying epineural patch harvested. Frozensections of epineurium and paraffin sections of epineural patch coveringthe DRG were stained with a monoclonal antibody for the presence ofneural growth factors GFAP, S100 and laminin and for the VEGF expressionusing immunofluorescence and immunoperoxidase staining respectively.

GFAP and S100 were expressed neither on epineurial sheath nor onepineural sheath patch covering DRG after laminectomy.

Laminin was constitutively expressed on both stained tissues.

VEGF was not detected within normal control epineurial sheath. Incontrast, in epineural sheaths harvested 72 hours after covering theDRG, numerous VEGF stained vessels were observed. See FIG. 17.

Degenerative spinal changes result in narrowing of the space availablefor neural structures. This in turn results in nerve ischemia (decreasein blood supply) and intra-neural changes such as fibrosis. It isbelieved that nerve ischemia leads to altered nerve function and pain.

As shown herein, the epineural sheath has angiogenic properties bydemonstrating increased expression of VEGF in the sheath substance 72hours after topical application onto the DRG.

The epineural sheath cam also be used to increase angioneogenesis(formation of new blood vessels) in chronically compressed lumbar nerveroots by topically applying the epineural sheath (gel, powder, etc) ontothe decompressed neural structures (nerve roots, DRG, nerves, dura).

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of repairing a nerve gap having a proximal nerve stump and adistal nerve stump in an individual in need thereof, comprising a)attaching an isolated, naturally-occurring epineural sheath to theproximal nerve stump and to the distal nerve stump, thereby producing anerve graft and b) maintaining the nerve graft under conditions in whichnerve tissue is regenerated between the proximal nerve stump and thedistal nerve stump, thereby repairing the nerve gap.
 2. The method ofclaim 1, comprising: a) attaching a flat epineural sheath to theproximal nerve stump and to the distal nerve stump, thereby producing anerve graft; and b) maintaining the nerve graft under conditions inwhich nerve tissue is regenerated between the proximal nerve stump andthe distal nerve stump, thereby repairing the nerve gap.
 3. The methodof claim 2, wherein the flat epineural sheath is attached to theproximal nerve stump using at least one suture and to the distal nervestump using at least one suture.
 4. The method of claim 2, wherein theflat epineural sheath is about 2 cm to about 10 cm in length and about 1mm to about 10 mm in width.
 5. The method of claim 2, wherein the nervegap is from about 1 mm to about 10 cm.
 6. A method of claim 1,comprising a) attaching an isolated, naturally occurring epineural tubeto the proximal nerve stump and to the distal nerve stump, therebyproducing a nerve graft and b) maintaining the nerve graft underconditions in which nerve tissue is regenerated between the proximalnerve stump and the distal nerve stump, thereby repairing the nerve gap.7. The method of claim 6, wherein the epineural tube is attached to theproximal nerve stump using at least one suture and to the distal nervestump using at least one suture.
 8. The method of claim 6, wherein theepineural tube is from about 1 cm to about 20 cm in length.
 9. Themethod of claim 6, wherein the nerve gap is from about 1 mm to about 20cm.
 10. The method of claim 6, wherein the epineural tube is anautologous epineural tube, an allogenic epineural tube, an isogenicepineural tube, a xenogenic epineural tube or a combination thereof. 11.A method of protecting neural tissue in an individual in need thereof,comprising a) attaching an isolated, naturally occurring epineuralsheath to the neural tissue, thereby covering the nerve tissue and b)maintaining the neural tissue under conditions in which the neuraltissue is isolated, thereby protecting the neural tissue.
 12. The methodof claim 11, wherein the neural tissue is injured neural tissue.
 13. Themethod of claim 12, wherein the injured neural tissue producesneuropathic pain in the individual.
 14. The method of claim 12, whereinthe injured neural tissue is compressed.
 15. The method of claim 11,wherein the neural regeneration occurs.
 16. The method of claim 11wherein the neural tissue is a dorsal root ganglion.
 17. The method ofclaim 11, wherein the epineural sheath is attached to the neural tissueusing at least one suture.
 18. The method of claim 11, wherein theepineural sheath is an autologous epineural sheath, an allogenicepineural sheath, an isogenic epineural sheath, a xenogenic sheath or acombination thereof.